Critical Factors for Successful Real-Time PCR - Gene Quantification

Critical Factors for Successful Real-Time PCR

Sample & Assay Technologies

Contents

1. Introduction

4

2. Detection of PCR products in real-time

5

2.1 SYBR Green

5

2.2 Fluorescently labeled sequence-specific probes

5

3. Methods in real-time PCR

8

3.1 Two-step and one-step RT-PCR

9

3.2 Multiplex PCR and RT-PCR

10

3.3 Real-time RT-PCR using cell lysates

10

3.4 Fast PCR and RT-PCR

11

3.5 Whole genome amplification

11

3.6 Whole transcriptome amplification

11

4. Basic terms used in real-time PCR

12

5. Quantification of target amounts

15

5.1 Quantification

16

5.2 Absolute quantification

16

5.3 Relative quantification

18

5.4 Controls

24

6. Guidelines for preparation of template, primers, and probes 6.1 Purification of nucleic acid templates

26

6.2 Handling and storing primers and probes

27

7. Guidelines for successful whole transcriptome amplification

2

26

28

7.1 WTA techniques

28

7.2 The importance of RNA quality

29

7.3 Working with degraded RNA

29

7.4 Amplification from low cell numbers

29

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Real-Time PCR Brochure 07/2010

Pagetitle optional

8. Guidelines for successful reverse transcription

31

8.1 Choice of RT primers

31

8.2 Effect of RT volume added to two-step RT-PCR

33

8.3 Reverse-transcription conditions for two-step RT-PCR

33

8.4 Reverse-transcription conditions for one-step RT-PCR

36

8.5 Removal of genomic DNA contamination

36

9. Guidelines for successful real-time PCR

37

9.1 Primer design

37

9.2 Reaction chemistry

42

9.3 SYBR Green-based detection in real-time PCR

43

9.4 Probe-based detection in real-time PCR

45

9.5 Real-time PCR with fast cycling

46

9.6 Real-time RT-PCR direct from cell lysates

49

9.7 Genomic DNA in real-time RT-PCR

52

10. Guidelines for successful multiplex real-time PCR

54

10.1 Guidelines for designing primers and probes

54

10.2 Guidelines for selecting appropriate reporter dyes and quenchers for the probes

54

10.3 Choice of PCR buffer

55

10.4 Guidelines for evaluating the performance of a multiplex assay

57

10.5 Guidelines for programming the real-time cycler

58

10.6 Guidelines for analyzing data from a multiplex assay

58

11. Guidelines for successful virus load quantitation

61

Selection Guide: QIAGEN products for real-time PCR and real-time RT-PCR

62



3

Pagetitle optional Introduction

1.0

1. Introduction Real-time PCR and RT-PCR are highly sensitive techniques enabling amplification and quantification of a specific nucleic acid sequence with detection of the PCR product in real time. Quantification of DNA, cDNA, or RNA targets can be easily achieved by determining the cycle when the PCR product can first be detected. This is in contrast with end-point detection in conventional PCR, which does not enable accurate quantification of nucleic acids. Real-time PCR is highly suited for a wide range of applications, such as gene expression analysis, determination of viral load, detection of genetically modified organisms (GMOs), SNP genotyping, and allelic discrimination. In this guide, we provide information on the basic principles of real-time PCR, terms used in real-time PCR, and factors influencing the performance of real-time PCR assays. We also cover the latest technologies in real-time PCR, including multiplex analysis, fast cycling, direct analysis of cell lysates, and whole transcriptome amplification. In addition, we describe the advantages and disadvantages of different reaction chemistries as well as provide answers to frequently asked questions and guidelines for successful results. Examples of the spectrum of research currently being carried out are also included. We extend our thanks to those who have contributed to this project and hope that it may provide a useful guide to successful real-time PCR for researchers everywhere.

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Pagetitle optional Detection of PCR products in real-time

2.0–2.2

2. Detection of PCR products in real-time Real-time PCR and RT-PCR allow accurate quantification of starting amounts of DNA, cDNA, and RNA targets. Fluorescence is measured during each cycle, which greatly increases the dynamic range of the reaction, since the amount of fluorescence is proportional to the amount of PCR product. PCR products can be detected using either fluorescent dyes that bind to double-stranded DNA or fluorescently labeled sequence-specific probes.

2.1 SYBR® Green The fluorescent dye SYBR Green I binds all double-stranded DNA molecules, emitting a fluorescent signal of a defined wavelength on binding (Figure 1). The excitation and emission maxima of SYBR Green I are at 494 nm and 521 nm, respectively, allowing use of the dye with any real-time cycler. Detection takes place in the extension step of real-time PCR. Signal intensity increases with increasing cycle number due to the accumulation of PCR product. Use of SYBR Green enables analysis of many different targets without having to synthesize target-specific labeled probes. However, nonspecific PCR products and primer–dimers will also contribute to the fluorescent signal. Therefore, high PCR specificity is required when using SYBR Green.

2.2 Fluorescently labeled sequence-specific probes

Figure 1. SYBR Green principle. Principle of SYBR Greenbased detection of PCR products in real-time PCR.

Fluorescently labeled probes provide a highly sensitive and specific method of detection, as only the desired PCR product is detected. However, PCR specificity is also important when using sequence-specific probes. Amplification artifacts such as nonspecific PCR products and primer–dimers may also be produced, which can result in reduced yields

A

of the desired PCR product. Competition between the specific product and reaction artifacts for reaction components can compromise assay sensitivity and efficiency. The following sections discuss different probe chemistries. B

2.2.1 TaqMan® probes TaqMan probes are sequence-specific oligonucleotide probes carrying a fluorophore and a quencher moiety (Figure 2). The fluorophore is attached at the 5' end of the probe and the quencher moiety is located at the 3' end. During the combined annealing/extension phase of PCR, the probe is cleaved by the 5' → 3' exonuclease activity of Taq DNA polymerase, separating the fluorophore and the quencher moiety. This results in detectable fluorescence that is proportional to the amount of accumulated PCR product. Examples of quencher moieties include TAMRA fluorescent dye and Black Hole Quencher® (BHQ®) nonfluorescent dyes.



Figure 2. TaqMan probe principle. A Both the TaqMan probe and the PCR primers anneal to the target sequence during the PCR annealing step. The proximity of the quencher to the fluorophore strongly reduces the fluorescence emitted by the fluorophore. B During the PCR extension step, Taq DNA polymerase extends the primer. When the enzyme reaches the probe, its 5' → 3' exonuclease activity cleaves the fluorophore from the probe. The fluorescent signal from the free fluorophore is measured. The signal is proportional to the amount of accumulated PCR product.

5

Detection of PCR products in real-time

2.2

2.2.2 FRET probes PCR with fluorescence resonance energy transfer (FRET) probes, such as LightCycler® hybridization probes, uses 2 labeled oligonucleotide probes that bind to the PCR product in a head-to-tail fashion (Figure 3). When the 2 probes bind, their fluorophores come into close proximity, allowing energy transfer from a donor fluorophore to an acceptor fluorophore. Therefore, fluorescence is detected during the annealing phase of PCR and is proportional to the amount of PCR product. FRET probes usually carry dyes that are only compatible with LightCycler and Rotor-Gene® instruments. As the FRET system uses 2 primers and 2 probes, good design of the primers and probes is critical for successful results. 2.2.3 Dyes used for fluorogenic probes in real-time PCR For real-time PCR with sequence-specific probes, various fluorescent dyes are available, each with its own excitation and emission maxima (Table 1 and Figure 4). The wide variety of dyes makes multiplex, real-time PCR Figure 3. FRET probe principle. A When not bound to the target sequence, no fluorescent signal from the acceptor fluorophore is detected. B During the PCR annealing step, both FRET probes hybridize to the target sequence. This brings the donor and acceptor fluorophores into close proximity, allowing energy transfer between the fluorophores and resulting in a fluorescent signal from the acceptor fluorophore that is detected. The amount of signal is proportional to the amount of target sequence, and is measured in real time to allow quantification of the amount of target sequence. C During the extension step of PCR, the probes are displaced from the target sequence and the acceptor fluorophore is no longer able to generate a fluorescent signal.

possible (detection of 2 or more amplicons in the same reaction), provided the dyes are compatible with the excitation and detection capabilities of the real-time cycler used, and the emission spectra of the chosen dyes are sufficiently distinct from one another. Therefore, when carrying out multiplex PCR, it is best practice to use dyes with the widest channel separation possible to avoid any potential signal crosstalk.

CAL Fluor Gold 540 (544) JOE (548) Yakima Yellow (548) VIC (552) HEX (553)

Alexa Fluor 350 (442) Marina Blue (460)

400

450

FAM(518) Alexa Fluor 488 (519) SYBR Green I (521) EvaGreen (530)

500

550

Cy3.5 (596) ROX (607) CAL Fluor Red 610 (610) Texas Red (615)

Cy5 (667) Alexa Fluor 647 (666) Quasar 670 (667)

CAL Fluor Red 635 (637) Bodipy 630/650 (640)

Bodipy TMR (574) NED (575)

600

Alexa Fluor 660 (690) Quasar 705 (705) Alexa Fluor 680 (702)

650

Figure 4. Emission maximum of selected reporter dyes. The emission maximum (nm) of selected reporter dyes are displayed in parentheses. Emission maximum may vary depending on buffer conditions. Other dyes with similar wavelengths may not be suitable for multiplex assays due to low fluorescence and/or stability.

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Detection of PCR products in real-time

2.2

Table 1. Dyes commonly used for quantitative, real-time PCR Dye

Excitation maximum (nm)

Emission maximum (nm)*

Fluorescein

490

513

Oregon Green®

492

517

FAM

494

518

SYBR Green I

494

521

TET

521

538

JOE

520

548

VIC®

538

552

Yakima Yellow®

526

552

HEX

535

553

Cy®3

552

570

Bodipy® TMR

544

574

NED

546

575

TAMRA

560

582

Cy3.5

588

604

ROX

587

607

Texas Red®

596

615

LightCycler Red 640 (LC640)

625

640

Bodipy 630/650

625

640

Alexa Fluor® 647

650

666

Cy5

643

667

Alexa Fluor 660

663

690

Cy5.5

683

707

* Emission spectra may vary depending on the buffer conditions.

?

My sample does not give a fluorescent signal. How can I decide whether this is because the PCR did not work or because the target is not expressed?

Use a control sample in which the gene of interest is definitely expressed or the target sequence is present. PCR products that span the region to be amplified in the real-time experiment can also be used as a positive control. Check by agarose gel electrophoresis that the amplification reaction was successful. The quality of the starting template and the integrity of the reagents can be determined by amplifying a housekeeping gene, such as GAPDH or HPRT, or by amplifying the cloned target sequence, either in the form of in vitro transcribed RNA or plasmid DNA.

?

Do I need to calibrate my real-time cycler in order to detect reporter dyes?

Some real-time cyclers require you to perform a calibration procedure for each reporter dye. Check whether the reporter dyes you selected for your assays are part of the standard set of dyes already calibrated on your instrument. If they are not, perform a calibration procedure for each dye before using them for the first time. For details about calibration, refer to the user manual supplied with your cycler.



7

Methods in real-time PCR

3.0

3. Methods in real-time PCR DNA template (e.g., genomic DNA or plasmid DNA) can be directly used as starting template in real-time PCR. Real-time PCR can be used to quantify genomic DNA, for example, in detection of bacterial DNA or GMOs, and can also be used for qualitative analysis, such as single nucleotide polymorphism (SNP) detection. RNA template is used for analysis of gene expression levels or viral load of RNA viruses. Real-time RT-PCR is carried out, where the RNA first needs to be transcribed into cDNA using a reverse transcriptase prior to PCR. Reverse transcriptases are enzymes generally derived from RNA-containing retroviruses. Real-time RT-PCR can be either a two-step or a one-step procedure, as described in section 3.1.

Application data Using real-time PCR for SNP genotyping SNP analysis involves the detection of single nucleotide changes using 2 probes labeled with different fluorophores. One probe is specific for the wild-type allele, the other for the mutant allele (Figure 5). Real-time PCR is highly suited for the detection of small sequence differences, such as SNPs and viral variants. A

ICAM5

B

BCAC3-29

Figure 5. Fast and reliable SNP genotyping. Real-time PCR with genomic DNA template and subsequent end-point allelic discrimination was carried out on the Applied Biosystems® 7500 Fast System using the QuantiFast ® Probe PCR +ROX Vial Kit and TaqMan probes for detecting SNPs in A ICAM5 (intercellular adhesion molecule) and B BCAC3-29. The allelic discrimination plots clearly indicate the homozygotes for each allele ( Allele X or Allele Y) and the heterozygotes ( Both). No template control is indicated by . The PCR run time was about 35 minutes. (Data kindly provided by Peter Schuermann, Department of Gynecology and Obstetrics, Hannover Medical School, Hannover, Germany.)

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3.1

3.1 Two-step and one-step RT-PCR Real-time RT-PCR can take place in a two-step or one-step reaction (Figure 6 and Table 2). With two-step RT-PCR, the RNA is first reverse transcribed into cDNA using oligo-dT primers, random oligomers, or gene-specific primers. An aliquot of the reverse-transcription reaction is then added to the real-time PCR. It is possible to choose between different types of RT primers, depending on experimental needs. Use of oligo-dT primers or random oligomers for reverse transcription means that several different transcripts can be analyzed by PCR from a single RT reaction. In addition, precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage. In one-step RT-PCR — also referred to as one-tube RT-PCR — both reverse transcription and real-time PCR take place in the same tube, with reverse transcription preceding PCR. This is possible due to specialized reaction chemistries and cycling protocols (see section 8.4, page 36). The fast procedure enables rapid processing of multiple samples and is easy to automate. The reduced number of handling steps results in high reproducibility from sample to sample and minimizes the risk of contamination since less manipulation is required. A Add RT-PCR master mix cDNA synthesis and PCR in 1 tube

RNA template

B Add RT master mix RNA template

Transfer cDNA aliquots to PCR master mix cDNA synthesis

PCR

Figure 6. Comparison of two-step and one-step RT-PCR. A In one-step RT-PCR, reverse transcription and PCR take place sequentially in the same tube. B In two-step RT-PCR, cDNA is synthesized in 1 tube, and aliquots of the cDNA are transferred to other tubes for PCR.

Table 2. Advantages of different RT-PCR procedures Procedure

Advantages

Two-step RT-PCR

Multiple PCRs from a single RT reaction Flexibility with RT primer choice Enables long-term storage of cDNA

One-step RT-PCR

Easy handling Fast procedure High reproducibility Low contamination risk



9


3.2–3.3

3.2 Multiplex PCR and RT-PCR In multiplex, real-time PCR, several genomic DNA targets are quantified simultaneously in the same reaction. Multiplex, real-time RT-PCR is a similar method, allowing simultaneous quantification of several RNA targets in the same reaction. The procedure can be performed either as two-step RT-PCR or as one-step RT-PCR (see section 3.1). Multiplex PCR and RT-PCR offer many advantages for applications such as gene expression analysis, viral load monitoring, and genotyping. The target gene(s) as well as an internal control are coamplified in the same reaction, eliminating the well-to-well variability that would occur if separate amplification reactions were carried out. The internal control can be either an endogenous gene that does not vary in expression between different samples (e.g., a housekeeping gene; see Table 7, page 24) or an exogenous nucleic acid. For viral load monitoring, the use of an exogenous nucleic acid as internal control allows the following parameters to be checked: the success of sample preparation, the absence of inhibitors, and the success of PCR. Multiplex analysis ensures high precision in relative gene quantification, where the amount of a target gene is normalized to the amount of a control reference gene. Quantification of multiple genes in a single reaction also reduces reagent costs, conserves precious sample material, and allows increased throughput. Multiplex PCR and RT-PCR are made possible by the use of sequence-specific probes that are each labeled with a distinct fluorescent dye and an appropriate quencher moiety. This means that the emission maxima of the dyes must be clearly separated and must not overlap with each other (see Figure 4 on page 6). In addition, reactions must be carried out on an appropriate real-time cycler that supports multiplex analysis (i.e., the excitation and detection of several non-overlapping dyes in the same well or tube). To view successful multiplex data from a wide range of real-time cyclers, visit www.qiagen.com/multiplex.

3.3 Real-time RT-PCR using cell lysates Real-time RT-PCR is an ideal tool for cell assays that require accurate analysis of gene expression, for example, in experiments to validate gene knockdown after transfection of siRNAs. However, high-throughput assays are difficult to achieve, since the purification of RNA from large numbers of cultured-cell samples involves both time and effort. This bottleneck can be overcome by eliminating the RNA purification steps and using cell lysates directly in real-time RT-PCR. However, the method for cell lysis needs to be carefully optimized so that the lysates provide similar performance in real-time RT-PCR as pure RNA templates. The method should preserve the gene expression profile and also prevent cellular and buffer components from interfering with amplification and detection. A reliable method for real-time RT-PCR direct from cells is presented at www.qiagen.com/FastLane.

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3.4–3.6

3.4 Fast PCR and RT-PCR Most new real-time cyclers are installed with thermal-cycling modules that provide high ramping rates (i.e., fast heating and cooling capacities). This technology shortens the time to switch from one temperature to another, allowing faster run times in real-time PCR. Further and more significant time savings can be achieved by reducing the duration of the denaturation, annealing, and extension steps in each PCR cycle as well as shortening the time required for activation of the hot-start DNA polymerase. When reducing these PCR parameters, care should be taken to avoid compromising the specificity and sensitivity of real-time PCR. Examples of fast PCR without compromising performance can be viewed for various different real-time cyclers at www.qiagen.com/fastPCR.

3.5 Whole genome amplification The small amount of genomic DNA in precious samples limits the number of real-time PCR analyses that can be carried out. This limitation can be overcome by whole genome amplification (WGA), a method which allows amplification of all genomic DNA targets in a sample. Unbiased and accurate amplification of whole genomes can be achieved with Multiple Displacement Amplification (MDA). For details, visit www.qiagen.com/WGA.

3.6 Whole transcriptome amplification Whole transcriptome amplification (WTA) allows amplification of entire transcriptomes from very small amounts of RNA, enabling unlimited analyses by real-time RT-PCR. WTA of RNA samples can be achieved by reverse transcription and cDNA ligation prior to MDA (see page 28).

Application data Single-base detection using quenched FRET assays Quenched FRET assays are similar to FRET assays except that the decrease in energy of the donor fluorophore is measured instead of the increase in energy of the acceptor fluorophore. FRET probes were designed to detect the single-base mutations H63D and S65C. The QuantiTect® Probe PCR Kit enabled highly sensitive detection of wild type and mutant sequences (Figure 7).

Wild type Fluorescence -dF/dT

5

H63D heterozygote H63D/S65C heterozygote

4 3 2 Threshold

1 0 –1 50

55

60


65

70

Figure 7. Sensitive detection of single-base mutations. Following real-time PCR using the QuantiTect Probe PCR Kit on the Rotor-Gene 3000, amplification products were subjected to melting curve analysis. (Data kindly provided by T. Kaiser, Corbett Research, Australia.)

75


11

Basic terms used in real-time PCR

4.0

4. Basic terms used in real-time PCR Before levels of nucleic acid target can be quantified in real-time PCR, the raw data must be analyzed and baseline and threshold values set. When different probes are used in a single experiment (e.g., when analyzing several genes in parallel or when using probes carrying different reporter dyes), the baseline and threshold settings must be adjusted for each probe. Furthermore, analysis of different PCR products from a single experiment using SYBR Green detection requires baseline and threshold adjustments for each individual assay. Basic terms used in data analysis are given below. For more information on data analysis, refer to the recommendations from the manufacturer of your real-time cycler. Data are displayed as sigmoidal-shaped amplification plots (when using a linear scale), in which the fluorescence is plotted against the number of cycles (Figure 8).

Fluorescence

Sample A Sample B

Log-linear phase Threshold Figure 8. Typical amplification plot. Amplification plots showing increases in fluorescence from 2 samples (A and B). Sample A contains a higher amount of starting template than sample B. The Y-axis is on a linear scale.

Baseline

0

10

0

20

30

40

Cycle number

Baseline: The baseline is the noise level in early cycles, typically measured between cycles 3 and 15, where there is no detectable increase in fluorescence due to amplification products. The number of cycles used to calculate the baseline can be changed and should be reduced if high template amounts are used or if the expression level of the target gene is high (Figure 9A). To set the baseline, view the fluorescence data in the linear scale amplification plot. Set the baseline so that growth of the amplification plot begins at a cycle number greater than the highest baseline cycle number (Figure 9B). The baseline needs to be set individually for each target sequence. The average fluorescence value detected within the early cycles is subtracted from the fluorescence value obtained from amplification products. Recent versions of software for various real-time cyclers allow automatic, optimized baseline settings for individual samples. B 24

24

20

20

16

16 ΔRn

ΔRn

A

12

12

8

8

4

4

-1

-1 1

7

18 25 13 Cycle number

31

37

1

7


31

37

Figure 9. Correct baseline and threshold settings are important for accurate quantification. A Amplification product becomes detectable within the baseline setting of cycles 6 to 15 and generates a wavy curve with the highest template amount. B Setting the baseline within cycles 6 to 13 eliminates the wavy curve. The threshold is set at the beginning of the detectable log-linear phase of amplification.

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4.0

Background: This refers to nonspecific fluorescence in the reaction, for example, due to inefficient quenching of the fluorophore or the presence of large amounts of double-stranded DNA template when using SYBR Green. The background component of the signal is mathematically removed by the software algorithm of the real-time cycler. Reporter signal: Fluorescent signal that is generated during real-time PCR by either SYBR Green or a fluorescently labeled sequence-specific probe. Normalized reporter signal (Rn): This is the emission intensity of the reporter dye divided by the emission intensity of the passive reference dye measured in each cycle. Passive reference dye: On some real-time cyclers, the fluorescent dye ROX serves as an internal reference for normalization of the fluorescent signal. It allows correction of well-to-well variation due to pipetting inaccuracies, well position, and fluorescence fluctuations. Its presence does not interfere with real-time PCR assays, since it is not involved in PCR and has an emission spectrum completely different from fluorescent dyes commonly used for probes. Threshold: The threshold is adjusted to a value above the background and significantly below the plateau of an amplification plot. It must be placed within the linear region of the amplification curve, which represents the detectable log-linear range of the PCR. The threshold value should be set within the logarithmic amplification plot view to enable easy identification of the log-linear phase of the PCR. If several targets are used in the real-time experiment, the threshold must be set for each target. Threshold cycle (CT) or crossing point (Cp): The cycle at which the amplification plot crosses the threshold (i.e., there is a significant detectable increase in fluorescence). CT can be a fractional number and allows calculation of the starting template amount (see section 5, page 15). ΔCT value: The ΔCT value describes the difference between the CT value of the target gene and the CT value of the corresponding endogenous reference gene, such as a housekeeping gene, and is used to normalize for the amount of template used: ΔCT = CT (target gene) – CT (endogenous reference gene) ΔΔCT value: The ΔΔCT value describes the difference between the average ΔCT value of the sample of interest (e.g., stimulated cells) and the average ΔCT value of a reference sample (e.g., unstimulated cells). The reference sample is also known as the calibrator sample and all other samples will be normalized to this when performing relative quantification: ΔΔCT = average ΔCT (sample of interest) – average ΔCT (reference sample) For more information on ΔCT and ΔΔCT, see section 5, page 15. Endogenous reference gene: This is a gene whose expression level should not differ between samples, such as a housekeeping gene (1). Comparing the CT value of a target gene with that of the endogenous reference gene allows normalization of the expression level of the target gene to the amount of input RNA or cDNA (see ΔCT value). The exact amount of template in the reaction is not determined. An endogenous reference gene corrects for possible RNA degradation or presence of inhibitors in the RNA sample, and for variation in RNA content, reverse-transcription efficiency, nucleic acid recovery, and sample handling. See page 24 for a list of housekeeping genes.



13


4.0

Internal control: This is a control sequence that is amplified in the same reaction as the target sequence and detected with a different probe (i.e., duplex PCR is carried out). An internal control is often used to rule out failure of amplification in cases where the target sequence is not detected. Calibrator sample: This is a reference sample used in relative quantification (e.g., RNA purified from a cell line or tissue) to which all other samples are compared to determine the relative expression level of a gene. The calibrator sample can be any sample, but is usually a control (e.g., an untreated sample or a sample from time zero of the experiment). Positive control: This is a control reaction using a known amount of template. A positive control is usually used to check that the primer set or primer–probe set works. No template control (NTC): This is a control reaction that contains all essential components of the amplification reaction except the template. This enables detection of contamination. No RT control: RNA preparations may contain residual genomic DNA, which may be detected in real-time RT-PCR if assays are not designed to detect and amplify RNA sequences only (see section 9.7, page 52). DNA contamination can be detected by performing a no RT control reaction in which no reverse transcriptase is added. Standard: This is a sample of known concentration or copy number used to construct a standard curve. Standard curve: To generate a standard curve, CT values/crossing points of different standard dilutions are plotted against the logarithm of input amount of standard material. The standard curve is commonly generated using a dilution series of at least 5 different concentrations of the standard. Each standard curve should be checked for validity, with the value for the slope falling between –3.3 to –3.8. Standards are ideally measured in triplicate for each concentration. Standards which give a slope differing greatly from these values should be discarded. Efficiency and slope: The slope of a standard curve provides an indication of the efficiency of the real-time PCR. A slope of –3.322 means that the PCR has an efficiency of 1, or 100%, and the amount of PCR product doubles during each cycle. A slope of less than –3.322 (e.g., –3.8) is indicative of a PCR efficiency 20 ng) in the RNA purification procedure should be avoided. However, total RNA purified with carrier RNA using the RNeasy Micro Kit or RNeasy Plus Micro Kit performs well in WTA using the QuantiTect Whole Transcriptome Kit.

7.3 Working with degraded RNA RNA from certain sources, such as formalin-fixed, paraffin-embedded (FFPE) tissue sections, may be degraded and not be suitable for WTA. For efficient RNA amplification with the QuantiTect Whole Transcriptome Kit, RNA should not be heavily degraded (i.e., RNA should be longer than 500 nucleotides). The method cannot be used to amplify RNAs substantially smaller than mRNA molecules, such as tRNAs or miRNAs.

7.4 Amplification from low cell numbers When carrying out WTA, it is important to consider both the amount of starting material (i.e., the number of cells or the amount of RNA) and the copy number of the transcripts of interest. Table 9 and Figure 21 show the relationship between the amount of starting material and transcript representation (note that this is only a guide: the number of transcripts per given amount of starting material can vary). In starting material where the copy number of a transcript is 10 or less



29

Guidelines for successful whole transcriptome amplification

High-copy transcripts Medium-copy transcripts Low-copy transcripts Mosaic transcripts

7.4

(highlighted in bold in Table 9), stochastic problems will occur (i.e., the unequal distribution of a very low number of transcripts in a highly dilute solution). This may result in underrepresentation of the low-copy transcript

Copy number

at the start of WTA. Special consideration should be given to mosaic 10,000,000

transcripts, which are derived from genes that are expressed only in a

1,000,000

subset of cells in tissues. Since these transcripts are not present in every

100,000

cell, they will not be accurately represented in low amounts of starting

10,000

material (i.e., 1–102 cells).

1,000

Reliable WTA depends on the copy number of the transcripts. If the

100

recommended amount of starting material (i.e., 10 ng intact RNA) is

10 1 0.01

amplified with the QuantiTect Whole Transcriptome Kit, all transcripts will 0.1

1 RNA (ng)

10

Figure 21. Transcript representation in different RNA amounts. Relationship between amount of starting RNA and copy number of high-copy, medium-copy, low-copy, and mosaic transcripts.

be accurately represented after amplification. 10 ng of RNA corresponds to about 500 cells, and even low-copy transcripts are well represented in this RNA amount. Using lower amounts of RNA or a very limited number of cells means that the starting material could have a partial representation or an absence of low-copy transcripts (Figure 22 provides an example). Table 9. Transcript representation in different cell amounts 103 cells*

Variation in CT values

102 cells†

10 cells‡

1 cell§

Amount of RNA (ng)

20

2

0.2

0.02

No. of high-copy transcripts

107

106

105

104

No. of medium-copy transcripts

10

10

4

10

102

No. of low-copy transcripts

3

10

10

2

10

1

No. of mosaics transcripts

102

10

1

0

5

3

* Complete representation of all transcripts. Figure 22. Stochastic problems when analyzing a low-copy transcript. Eight replicate RNA samples (1 ng each) were amplified using the QuantiTect Whole Transcriptome Kit. Real-time PCR analysis of NFκB transcript was then performed using 10 ng amplified cDNA and the QuantiFast Probe PCR Kit. The resulting CT values were in the range of 30–32.5. This significant variation occurred because the stochastic variation in low-copy NFκB transcript in the replicate RNA samples is amplified, resulting in widely differing amounts of NFκB cDNA.

30

†

Stochastic problems for mosaic transcripts.

‡

Stochastic problems for low-copy and mosaic transcripts.

§

Stochastic problems for low-copy transcripts and loss of mosaic transcripts.

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Guidelines for successful reverse transcription

8.0–8.1

8. Guidelines for successful reverse transcription When performing real-time RT-PCR, the primers and the enzyme for reverse transcription must be carefully chosen. The primers should allow reverse transcription of all targets of interest, and the reverse transcriptase should yield cDNA amounts that accurately represent the original RNA amounts to ensure accurate quantification. In addition, the effects of the components of the RT reaction on subsequent real-time PCR must be minimized.

8.1 Choice of RT primers The choice of primers for reverse transcription depends on whether one-step or two-step RT-PCR is being carried out (see section 3.1, page 9). In one-step RT-PCR, the downstream PCR primer is also the primer for reverse transcription. Therefore, one-step RT-PCR is always performed with gene-specific primers. In two-step RT-PCR, 3 types of primers, and mixtures thereof, can be used for reverse transcription: oligo-dT primers (13–18mers), random oligomers (such as hexamers, octamers, or nonamers), or gene-specific primers (Table 10). If oligo-dT primers are used, only mRNAs will be reverse transcribed starting from the poly-A tail at the 3' end. Random oligomers will enable reverse transcription from the entire RNA population, including ribosomal RNA, transfer RNA, and small nuclear RNAs. Since reverse transcription is initiated from several positions within the RNA molecule, this will lead to relatively short cDNA molecules. In comparison, gene-specific primers allow reverse transcription of a specific transcript. Table 10. Suitability of primer types for RT-PCR Application

Recommended type of primer

RT-PCR of specific transcript:

Gene-specific primer gives highest selectivity and only the RNA molecule of choice will be reverse transcribed

RT-PCR of long amplicon:

Oligo-dT or gene-specific primers

RT-PCR of an amplicon within long transcript:

Gene-specific primers, random oligomers, or a mixture of oligo-dT primers and random nonamers (see section 8.1.1) are recommended so that cDNA covering the complete transcript is produced

8.1.1 Universal priming method for real-time two-step RT-PCR A universal priming method for the RT step of real-time two-step RT-PCR should allow amplification and detection of any PCR product regardless of transcript length and amplicon position, and achieve this with high sensitivity and reproducibility. To determine the optimal primers for reverse transcription, 2 regions within a 10-kb transcript were analyzed by real-time two-step RT-PCR. Each amplicon was approximately 150 bp in length. For the amplicon 2 kb away from the 3' end of the transcript, the lowest CT value was obtained using either oligo-dT primers alone or a mixture of oligo-dT primers and random nonamers (Figure 23A). However, when the amplicon was 6 kb away from the 3' end of the transcript, random nonamers provided the lowest CT value, closely followed by the mixture of oligo-dT primers and random nonamers (Figure 23B). The use of oligo-dT primers alone resulted in a much higher CT value, indicating transcript detection was significantly less sensitive. Random nonamers proved to be better than random hexamers or dodecamers (data not shown; the performance of random nonamers varies depending on the reverse transcriptase used).



31


8.1

This relationship between choice of RT primers and sensitivity of real-time RT-PCR has been observed with many different transcripts and RT-PCR systems. Therefore, when quantifying several targets from one RNA population, we recommend using a mixture of oligo-dT primers and random nonamers for reverse transcription. The QuantiTect Reverse Transcription Kit, which is specially designed to synthesize cDNA for use in real-time PCR, contains an optimized primer mix consisting of a unique blend of oligo-dT and random primers (Figure 24). Amplicon 2 kb from 3' end of template

A

3.0

3.5

Oligo-dT [1 μM] Oligo-dT+(N)9 [1 μM/10 μM] (N)9 [10 μM]

Oligo-dT [1 μM] Oligo-dT+(N)9 [1 μM/10 μM] (N)9 [10 μM]

2.5 2.0

ΔRn

2.5 ΔRn

Amplicon 6 kb from 3' end of template

B

1.5

1.5 1.0 0.5

0.5

0

0 15

20

25 30 Cycle number

35

CAP

40

AAA

15

CAP

20

25 30 Cycle number

35

40

AAA

Figure 23. Effect of RT primer choice on RT-PCR. Two-step RT-PCR was carried out using the QuantiTect SYBR Green PCR Kit and the primer combinations shown. The amplicon was A 2 kb from the 3' end or B 6 kb from the 3' end of the template RNA.

Application data Highly sensitive real-time two-step RT-PCR In contrast with other reverse transcriptases, the QuantiTect Reverse Transcription Kit provides higher yields of cDNA from any transcript region, allowing high sensitivity in real-time two-step RT-PCR. 7

QIAGEN Supplier R Supplier I

6

RFU

5 4 3 2 1 0 10

15

20

25

30

35

40

Figure 24. Sensitive detection of a target at the 5' region of a 12.5-kb transcript. Total RNA from mouse testis was reverse transcribed using the QuantiTect Reverse Transcription Kit or reverse transcriptases from Supplier I and Supplier R. Identical volumes of triplicate RT reactions were used in real-time PCR on the LightCycler system to analyze a target located at the 5' region of the dystrophin gene (about 12.5 kb upstream of the poly-A site). The error bars show the standard deviation for each set of triplicates. Compared with the other 2 kits, the QuantiTect Reverse Transcription Kit generated much higher amounts of cDNA (indicated by the lower CT values) and provided greater reproducibility in real-time RT-PCR (indicated by the smaller error bars). (Data kindly provided by Dr. Andrej-Nikolai Spiess, Department of Molecular Andrology, University Hospital Hamburg, Germany.)

Cycle

32

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8.2–8.3

3.5

In two-step RT-PCR, the addition of the completed reverse-transcription

2.5

reaction to the subsequent amplification reaction transfers not only cDNA template, but also salts, dNTPs, and RT enzyme. The RT reaction buffer, which has a different salt composition to that of the real-time PCR buffer,

Fluorescence

8.2 Effect of RT volume added to two-step RT-PCR

1.5 0.5

can adversely affect real-time PCR performance. However, if the RT reaction

0.0

forms 10% or less of the final real-time PCR volume, performance will not

–0.5 10

be significantly affected (Figure 25). Use of 3 µl of RT reaction in a 20 µl


15

PCR (i.e., 15% of the final volume) can lead to significant inhibition of real-time PCR (Figure 25). We recommend testing dilutions of the RT reaction in real-time PCR to check the linearity of the assay. This helps to eliminate any inhibitory effects of the RT reaction mix that might affect

35

40

Figure 25. Inhibition of real-time PCR by addition of RT reaction. Real-time PCR (20 µl volume) was carried out using plasmid DNA as template. The volumes of RT reaction (without template RNA) indicated above were added to the PCR to determine their effect on amplification.

accurate transcript quantification. A 4.000

8.3 Reverse-transcription conditions for two-step RT-PCR

3.500

The QuantiTect Reverse Transcription Kit provides efficient and sensitive

10 ng 3.000

Transcriptase, which is a unique mix consisting of Omniscript® and Sensiscript Reverse Transcriptases. Omniscript Reverse Transriptase is

ΔRn

reverse transcription of any template with Quantiscript® Reverse

1 ng

2.500

100 pg

2.000

designed for reverse transcription of RNA amounts greater than 50 ng,

1.500

and Sensiscript Reverse Transcriptase is optimized for use with very small amounts of RNA (